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At least 757 records · Page 42Linked to original sources
PubMed1997

Production and characterization of recombinant active mouse gelatinase B from eukaryotic cells and in vivo effects after intravenous administration.

Gelatinase B is a matrix metalloproteinase involved in tissue remodelling. When mouse cells are triggered in vitro with interleukin-1, bacterial endotoxin, virus-mimicking double-stranded RNA or cytokine inducers, they produce gelatinase B. To test the effects of gelatinase B in vivo, the enzyme was expressed in Chinese hamster ovary (CHO) cells. Hybrid genomic DNA-cDNA constructs under the control of two constitutive viral promoters were generated by PCR-mediated exon amplification. In vitro transcription and translation of the mRNA in reticulocyte lysate yielded the correct 79-kDa protein, and expression in CHO cells resulted in an intact glycosylated 110-kDa gelatinase B which was enzymically active. However, the production yields of recombinant enzyme from 50 tested clones were low and cell-culture supernatants contained significant amounts of copurifiable endogenous CHO gelatinase B. Therefore, the enzyme was expressed in the yeast Pichia pastoris. Recombinant proenzyme was secreted and recovered from the yeast culture medium at 10 mg/l. Amino-terminal sequence analysis indicated that affinity purification of the recombinant protein on gelatin-Sepharose yielded the expected N-glycosylated proenzyme form (110 kDa) in addition to an amino-terminally truncated unglycosylated variant (69 kDa). Both forms had gelatinolytic activity on zymography. The recombinant mouse gelatinase B was used to determine its pharmacokinetics and its haematological effects in vivo. After intravenous injection in rabbits, gelatinase B disappeared from the circulation within 6 h. In addition to a transient leukopenia, we observed a rapid increase in leukocytosis, which indicates that gelatinase B might be a factor involved in the desorption of adherent leukocytes from the vascular bed and in the release of leukocytes from the bone marrow. Gelatinase B secretion and activation might well be one of the crucial molecular mechanisms explaining leukocytosis which is associated with infections and almost all types of inflammation.

Animals↗
PubMed1974

Pancreatic ribonuclease: enzymic and physiological properties of a cross-linked dimer.

Monomeric ribonuclease A has very low activity toward typically double-stranded RNA's; the dimeric form of ribonuclease A obtained by cross linking the enzyme by dimethyl suberimidate has more than 78 times the activity of the monomer toward polyadenylate . polyuridylate and 440 times the activity of the monomer toward the double-stranded RNA of a virus from Penicillium chrysogenum. The half-life of the dimer in the bloodstream of the rat is 12 times that of the mononmer.

Animals↗
PubMed2000

Molecular epidemiology of metronidazole resistance in a population of Trichomonas vaginalis clinical isolates.

Trichomonas vaginalis, the causative agent for human trichomoniasis, is a problematic sexually transmitted disease mainly in women, where it may be asymptomatic or cause severe vaginitis and cervicitis. Despite its high prevalence, the genetic variability and drug resistance characteristics of this organism are poorly understood. To address these issues, genetic analyses were performed on 109 clinical isolates using three approaches. First, two internal transcribed spacer (ITS) regions flanking the 5.8S subunit of the ribosomal DNA gene were sequenced. The only variation was a point mutation at nucleotide position 66 of the ITS1 region found in 16 isolates (14.7%). Second, the presence of a 5.5-kb double-stranded RNA T. vaginalis virus (TVV) was assessed. TVV was detected in 55 isolates (50%). Finally, a phylogenetic analysis was performed based on random amplified polymorphic DNA data. The resulting phylogeny indicated at least two distinct lineages that correlate with the presence of TVV. A band-sharing index indicating relatedness was created for different groups of isolates. It demonstrated that isolates harboring the virus are significantly more closely related to each other than to the rest of the population, and it indicated a high level of relatedness among isolates with in vitro metronidazole resistance. This finding is consistent with the hypothesis that drug resistance to T. vaginalis resulted from a single or very few mutational events. Permutation tests and nonparametric analyses showed associations between metronidazole resistance and phylogeny, the ITS mutation, and TVV presence. These results suggest the existence of genetic markers with clinical implications for T. vaginalis infections.

Animals↗
PubMed2004

[Outlooks for using the interferon inducers in the treatment and prevention of influenza and acute respiratory viral diseases].

The methods of treatment by using the stimulators of the non-specific resistance system have been growing even more topical. Drugs based on interferon (INF) and its inducers belong to the group. IFN is of key importance in the regulation of antiviral immunity. The use of IFN inducers can be referred to as the most promising approach to enhancing and activating the body resistance mechanisms. The advantages of IFN inducers before IFN drugs, a wide spectrum of antiviral effects, compatibility with many pharmaceuticals, good tolerability by patients and effectiveness in different administration modes served as a basis for designing a new class of antiviral drugs--IFN inducers. A lot of experimental medical-and-biological research was made to evaluate the efficiency of IFN inducers in influenza. They were shown to possess a pronounced therapeutic-and-preventive action in experiment and clinical trials. Synthetic and natural dsRNA belong to IFN inducers. Multiple research made in cell culture and with experimental animals demonstrated a high interferon-inducing and antiviral activity of double-stranded RNA against influenza virus. A number of dsRNA-based IFN inducers are permitted for clinical use. Data obtained in clinical trials of such inducers are indicative of their pronounced therapeutic-and-clinical effect. Hence, the IFN inducers having different structures and origins can be regarded as promising in the treatment and prevention of influenza and acute respiratory viral diseases.

Acute Disease↗
PubMed1991

Interferon-gamma-inducible regulation of the human invariant chain gene.

Interferon-gamma (IFN-gamma) regulates a variety of immunoregulatory functions through the induction of a specific set of IFN-gamma response genes. This includes the invariant chain associated with the major histocompatibility complex class II molecules. To investigate the mechanism involved in the invariant chain (In) response to IFN-gamma we constructed chloramphenicol acetyltransferase (CAT) hybrid genes in which the CAT gene is under the control of the In promoter. The glioblastoma cell line, U-373 MG, transfected with a CAT construct having the In promoter sequence -790 to +1 bp showed over 3-fold increased CAT activity when treated with IFN-gamma indicating that this region confers IFN-gamma responsiveness to the CAT gene. The IFN-gamma response element in the promoter was further sublocalized to the region -120 to -61 base pairs (bp). This region contains homology to the interferon-stimulated response elements identified in other IFN responsive genes. By gel shift analyses, an IFN-gamma-induced sequence-specific DNA-binding factor was identified. This induced complex binds to an oligonucleotide corresponding to -107 to -79 bp of the In promoter. Mutations of this binding site at -94 and -92 bp drastically decreased binding of the constitutive and IFN-gamma-induced complexes. This IFN-gamma induced factor also binds to an oligonucleotide corresponding to -91 to -62 bp of the interferon-beta (IFN-beta) gene promoter, a region necessary for the induction of the IFN-beta gene by virus and double-stranded RNA. This binding specificity is characteristic of a family of DNA binding factors that bind both the interferon-stimulated response elements and the IFN-beta gene promoter.

Base Sequence↗
PubMed1982

2'-5'-Linked oligo(adenylic acid) and its analogs. A new class of inhibitors of mRNA methylation.

Most eukaryotic cellular and viral mRNAs have a blocked, methylated 5' terminus, commonly referred to as a "cap." The 5'-terminal 7-methyl-G in mRNAs is essential for their efficient translation in vitro and may also protect mRNAs against exonucleases. We have explored whether enzymes which synthesize 5' caps of mRNAs are targets for antiviral agents. We have reported earlier that the 5'-triphosphate of a broad spectrum antiviral agent, Ribavirin, and the 5'-mono- and triphosphates of 2'-5'-linked oligo(adenylic acid) (2-5A) synthesized by interferon-treated cells on exposure to double-stranded RNA or EMC virus inhibit in vitro methylation of unmethylated vaccinia RNA by a crude mRNA methylating enzyme system from vaccinia virus. We report here that although 2-5A inhibits both purified vaccinia viral and cellular mRNA (guanine-7-)-methyltransferases at micromolar concentrations, the 3'-O-methylated analogs of 2-5A, methylated in the 3'-terminal-OH or methylated at all three 3'-OH groups and with varying numbers of phosphate groups, specifically inhibited the viral enzyme at submicromolar concentrations. The inhibition is noncompetitive with respect to S-adenosylmethionine, but competitive with respect to mRNA substrate. These compounds are at least 10 times more active than 2-5A. A specific inhibitor of viral mRNA methylation heretofore has not been reported.

Adenine Nucleotides↗
PubMed2004

RNA interference as a new biotechnological tool for the control of virus diseases in plants.

RNA silencing occurs in a wide variety of organisms, including protozoa, fungi, plants and animals and involves recognition of a target RNA and initiation of a sequence-specific RNA degradation pathway in the cytoplasm. In the last few years, there have been considerable advances in our understanding of post-transcriptional gene silencing (PTGS). This mechanism is conceived as a natural antiviral defense system in plants that is activated as a response to double-stranded RNA (dsRNA) formed during virus replication. To develop new approaches for plant protection against virus diseases based on PTGS we have expanded previous findings on RNA interference (RNAi) in animals by using dsRNA to specifically interfere with virus infection in plants. This approach differs from strategies based on transgenic expression of RNAs but still relies on PTGS as a means to achieve pathogen-derived resistance (PDR). Our findings suggest that exogenously supplied dsRNA could form the basis for the development of an environmentally safe, new biotechnological tool aimed at protecting crops against virus diseases, provided that some limitations of the current status of the approach could be overcome.

Biotechnology↗
PubMed1979

The effect of ribonuclease on the replicative forms of Sindbis virus RNA.

Three species of double-stranded RNA, designated RF I, RF II, and RF III in order of decreasing size (25), are produced by ribonuclease treatment of extracts of chicken embryo cells infected for 6 hours with Sindbis virus. Only one class of replicative form RNA is present in extracts not treated with ribonuclease; this class contains some molecules which can be enzymatically cleaved to produce the other two replicative forms. At a low level of enzyme (0.001 microgram/ml) the major species obtained was RF I, the replicative form of the genome. When the enzyme concentration was increased 10-, 100-, and 1000-fold, there was a progressive increase in the proportions of RF's II and III and a concomitant decrease in the proportion of RF I. The generation of RF's II and III by nuclease resulted in the ratio expected for these two species if they are produced by cleavage of RF I-like molecules. In preparations of isolated double-stranded RNA, only RF I and replicative intermediate RNA were present. Mild nuclease treatment of these preparations converted the replicative intermediates primarily to RF I. Higher enzyme levels generated greater proportions of RF II and RF III, but RF I-like molecules were the major source for these increased proportions. Treatment of the isolated naturally occurring replicative form with 0.01 microgram of ribonuclease per ml cleaved some molecules migrating as RF I during gel electrophoresis into molecules which migrated as RF II and RF III.

Animals↗
PubMed1983

Differential antiviral effects of interferon in three murine cell lines.

Infectious leukemia virus production by two chronically infected NIH/MOL lines was strongly inhibited by interferon treatment of the cells. The corresponding degree of inhibition in JLSV-11 cells was much lower. Multiplication of encephalomyocarditis virus in all three cell lines was barely affected by interferon treatment. Replication of vesicular stomatitis virus, on the other hand, was highly sensitive to interferon in the JLSV-11 line and in one NIH/MOL line but was practically insensitive in the other NIH/MOL line. Anticellular actions of interferon were more pronounced in the JLSV-11 line than in the others. In response to interferon treatment, 2',5'-oligoadenylate synthetase activity was induced to a high level in JLSV-11 cells and to lower levels in the NIH/MOL lines. We failed to detect any 2',5'-oligoadenylate-dependent endonuclease activity in extracts of these cells. Double-stranded RNA-dependent protein kinase activity was present in extracts of interferon-treated NIH/MOL cells, but it was barely detectable in extracts of interferon-treated JLSV-11 cells. The above studies demonstrated that interferon could differentially affect the replication of three different viruses in three different cell lines, including two seemingly identical NIH/MOL lines, and that certain tentative conclusions can be drawn regarding the roles of different interferon-inducible enzyme markers in the different antiviral actions of interferons.

2',5'-Oligoadenylate Synthetase↗
PubMed1984

Two new double-stranded RNA molecules showing non-mendelian inheritance and heat inducibility in Saccharomyces cerevisiae.

Certain strains of Saccharomyces cerevisiae were found to have a complex nuclear defect (designated clo-) that makes cells unable to maintain some L-B and some L-C double-stranded RNAs at 25 degrees C. The clo- strains were not defective in maintenance of L-A, M1, or M2 double-stranded RNAs. Most clo-strains lacking L and M carry small amounts of two double-stranded RNA species intermediate in size between L and M and denoted T (2.7 kilobase pairs) and W (2.25 kilobase pairs). Some strains carry both T and W, some carry neither, and some carry only W; no strains carrying only T have been found. Both T and W show 4+:0 segregation in meiosis and efficient transmission by cytoplasmic mixing (cytoduction), indicating that they are non-Mendelian genetic elements. T and W do not cross-hybridize with each other or with L-A, L-B, L-C, M1, M2, or chromosomal DNA. T and W are apparently distinct from other known non-Mendelian genetic elements (2mu DNA, [rho], [psi], 20S RNA, [URE3]). In most strains the copy number of both T and W is increased about 10-fold by the growth of cells at 37 degrees C. This heat inducibility of T and W is under control of a cytoplasmic gene. T and W double-stranded RNAs are not found in a purified L-containing virus-like particle preparation from a strain containing L-B, T, and W double-stranded RNAs. The role, if any, of T or W in the killer systems is not known.

Genes, Fungal↗
PubMed1996

Secondary structure of the panhandle RNA of influenza virus A studied by NMR spectroscopy.

The double-stranded panhandle structure of the influenza virus RNA is important for replication, transcription and packaging into the virion of the virion RNA. The solution structure of a 34 nt RNA which contains the conserved panhandle sequences has been investigated by one- and two-dimensional NMR spectroscopy. The partially complementary 5'- and 3'-ends of the RNA form a double helical structure which is, on average, close to A-form. The stem contains bulges at nucleotides A10, A12 and C26. In between these bulges, C11 and G25 form a Watson-Crick base pair. The structural features of the panhandle provide a framework for the explanation of mutational analysis and for a better understanding of RNA-polymerase interactions.

Influenza A virus↗
PubMed2004

Genome assembly and particle maturation of the birnavirus infectious pancreatic necrosis virus.

In this study, we have analyzed the morphogenesis of the birnavirus infectious pancreatic necrosis virus throughout the infective cycle in CHSE-214 cells by using a native agarose electrophoresis system. Two types of viral particles (designated A and B) were identified, isolated, and characterized both molecularly and biologically. Together, our results are consistent with a model of morphogenesis in which the genomic double-stranded RNA is immediately assembled, after synthesis, into a large (66-nm diameter) and uninfectious particle A, where the capsid is composed of both mature and immature viral polypeptides. Upon maturation, particles A yield particles B through the proteolytic cleavage of most of the remaining viral precursors within the capsid, the compaction of the particle (60-nm diameter), and the acquisition of infectivity. These studies will provide the foundation for further analyses of birnavirus particle assembly and RNA replication.

Animals↗
PubMed1998

Rubella virus replication complexes are virus-modified lysosomes.

Replication complexes are membrane-bound cytoplasmic vacuoles involved in rubella virus (RV) replication. These structures can be identified by their characteristic morphology at the electron microscopy (EM) level and by their association with double-stranded (ds) RNA in immunogold labeling EM studies. Although these virus-induced structures bear some resemblance to lysosomes, their exact nature and origin are unknown. In this study, the localization of two lysosomal markers, lysosomal-associated membrane protein (Lamp-1) and acid phosphatase, relative to the replication complexes was examined by light and electron microscopy. Confocal microscopy using antibodies to dsRNA and Lamp-1 showed colocalization of these two markers in the cytoplasm of RV-infected cells. Immunogold labeling EM studies using antibodies to Lamp-1 confirmed that Lamp-1 was associated with RV replication complexes. EM histochemical studies demonstrated the presence of acid phosphatase in the vacuoles of RV replication complexes. Taken together, these studies show that RV replication complexes are virus-modified lysosomes.

Acid Phosphatase↗
PubMed1994

Virus-like particles in Eimeria nieschulzi are associated with multiple RNA segments.

RNA preparations from sporulated oocysts of Eimeria nieschulzi were found to contain 2 double-stranded RNA segments of 5.0 kb and 5.7 kb that were not present in other species of Eimeria. Treatment of crude lysates with RNase A revealed that in addition to these two segments, 3 other segments of 0.57 kb, 0.72 kb and 11.5 kb were protected from digestion, suggesting that they were enclosed within particles. Virus-like particles with a diameter of approximately 39 nm were purified by caesium chloride buoyant density centrifugation. Four of the five RNA segments copurified with these particles. In keeping with the nomenclature generally adopted for protozoan viruses, we have named this new isolate ENV 1. The largest RNA segment does not cosediment with ENV 1 particles and may be derived from another RNA-protein complex that is unstable under the conditions used. The particle size and genome structure of ENV 1 both differ from that of the Eimeria stiedae virus (ESV), which is the only other virus to have been isolated from Eimeria to date. Short cDNA clones derived from ENV 1 show significant homology to a region of the Leishmania virus (LRV 1) genome that encodes an RNA-dependent RNA polymerase. The polymerase sequences from ENV 1 and LRV 1 are more closely related to each other than to any other protein sequences in the GenEMBL Database. This raises intriguing questions about the origins of the two viruses, since Eimeria and Leishmania normally infect different hosts and also show different cell tropisms within these hosts.

Amino Acid Sequence↗
PubMed1979

Specific protein phosphorylation in interferon-treated uninfected and virus-infected mouse L929 cells: enhancement by double-stranded RNA.

The enhanced phosphorylation of specific protein(s) observed in extracts from interferon-treated cells (in the presence of ATP and double-stranded [ds] RNA) was also seen in intact mouse L929 cells upon treatment with dsRNA, polyriboinosinic.polyribocytidylic acid [poly(rI.rC)] or reovirus dsRNA, using 32Pi as radiolabel. Labeling of a 65,000-dalton protein(s) with 32P was greatly increased in interferon-treated cells in the presence of added dsRNA, suggesting that the expression in vivo of the kinase activity involved is regulated by dsRNA. This was used as a test system to investigate whether the activity of interferon-induced enzyme(s) is stimulated following virus infection, possibly owing to the accumulation of dsRNA. No obvious increase in 32P-labeling of 65,000-dalton protein(s) was observed upon infection of interferon-treated cells with mengovirus or vesicular stomatitis virus. A basal level of 32P-labeling of the 65,000-dalton protein(s) was detected in interferon-treated cells in the absence of added dsRNA, indicating a basal level of expression of the kinase activity involved. The possible implications of these results are discussed.

Dactinomycin↗
PubMed1986

Characterisation of orbiviruses of the Kemerovo serogroup: comparison of protein and RNA profiles.

Nine viruses of the Kemerovo serogroup (orbivirus genus; family, Reoviridae) isolated from seabird ticks (Ixodes uriae and Ornithodoros maritimus) from eight different geographical locations (four from Scotland, two from Morocco, one from Eire, one from England, one from the Faeroes Islands) were examined. All nine viruses produced unique RNA electropherotypes but showed a 2 : 4 : 3 : 1 grouping of the ten double-stranded RNA segments. The virus-induced proteins were labelled with 35S-methionine in a Vero cell line. Seven of the viruses isolated from Ixodes uriae produced similar profiles of nine major polypeptides. The remaining two viruses, isolated from Ornithodoros maritimus from Morocco, produced profiles in which only five major viral proteins could be readily detected, four of which had similar molecular weights to proteins apparent in the other isolates.

Animals↗